Novel Fosfomycin Resistance Mechanism in Pseudomonas entomophila Due to Atypical Pho Regulon Control of GlpT

Abstract

Background/Objectives:Pseudomonas entomophila is a ubiquitous bacterium capable of killing insects of different orders and has become a model for host-pathogen studies and a promising tool for biological pest control. In the human pathogen Pseudomonas aeruginosa, spontaneous resistance to fosfomycin arises almost exclusively from mutations in the glycerol-3-phosphate transporter (GlpT), the drug's sole entry route in this species. Here, we investigated whether this specificity is conserved in P. entomophila, as it could provide a valuable marker system for studying mutation rates and spectra and for selection in genetic engineering. Methods: We isolated 16 independent spontaneous fosfomycin-resistant mutants in P. entomophila, and studied the genetic basis of the resistance using a combination of sequencing, phenotyping and computational approaches. Results: We only found two mutants without alterations in glpT or any of its known regulatory elements. Whole-genome sequencing revealed unique inactivating mutations in phoU, a key regulator of the phosphate starvation (Pho) regulon. Computational analyses identified a PhoB binding site in the glpT promoter, and experiments showed that phoU inactivation reduced glpT expression nearly 20-fold. While placing a sugar-phosphate transporter under the Pho regulon may seem advantageous, bioinformatic analysis shows this configuration is atypical among pseudomonads. Conclusions: This atypical Pho regulon control of GlpT probably reflects the peculiarities of P. entomophila's habitat and lifestyle; highlighting how readily regulatory evolution can lead to the rapid divergence of resistance mechanisms, even among closely related species.

Keywords: GlpT; Pho regulon; fosfomycin resistance; mutation rate; promoter evolution.

Figure 1

Genetic features of spontaneous fosfomycin-resistant P. entomophila isolates: (A) Distribution of mutation types as detailed in Table 1, categorized by deletions, insertions, missense, nonsense, and absence of mutations. Different shades of the same color represent distinct mutations within the same category; (B) Mutation mapping along the open reading frames of the glpT and phoU genes. Bar height indicates the abundance of mutations at each position. As shown in Table 1, all mutations occur only once, except the deletion at codon 423 (which appears five times. Note that mutations are scattered along the glpT sequence, consistent with their presumed loss-of-function nature. The colors correspond to those in panel (A).

Figure 2

Phenotypic features of spontaneous fosfomycin-resistant P. entomophila isolates: (A) Relative expression levels of the glpT gene in the two mutants with unique loss-of-function mutations in phoU and the wild-type strain, as measured by qRT-PCR. Colors correspond to those in Figure 1A; (B) Minimum inhibitory concentration (MIC) values in the same two phoU mutants and the wild-type strain (average of three replicates, with no observed deviation among them). For reference, a glpT mutant with a nonsense mutation (Q372*) is included, representing the upper bound for resistance when GlpT-mediated fosfomycin transport is absent. Note the y-axis logarithmic scale. The colors correspond to those in Figure 1A. Symbols follow standard convention: deletion (Δ), stop codon (*).

Figure 3

Computational analysis of glpT regulation across pseudomonads and related species: the neighbor-joining phylogenetic tree on the (left) is based on pairwise distances among the genomes analyzed in this study (Table S1). The heatmap on the (right) illustrates the abundance and distribution of transcription factor binding sites predicted upstream of the glpT gene across the analyzed species. Black rows indicate species lacking a clear glpT homolog. The colors indicate the frequency of each binding site across species, with dark purple representing the highest occurrence, light blue representing a single occurrence, and white representing no hits. The labels of P. entomophila and phoB are highlighted in red for reference.